The goal of this research is to characterize expression and assembly of Treponema denticola (Td) surface proteins that mediate interactions with host tissue, thereby gaining insight into mechanisms of periodontal disease. We focus on analysis of two Td protein complexes that directly affect host cells: the oligomeric Msp protein and the PrtP lipoprotein protease complex. The overall hypothesis is that Msp and PrtP are major contributors to Td cytopathic behavior in periodontal disease. Expression of Msp and the PrtP complex are interdependent. To understand their roles in microbe-host interactions, we must understand how these molecules are expressed and interact in the spirochete. We propose genetic analysis of structural features and interactions of these complexes and their components. Our approach is to define the mechanisms of interdependence between Msp and the PrtP complex in order to characterize their roles in microbe-host interactions. Aim 1: Determine requirements for assembly and activity of the PrtP complex. We will construct a series of specific mutations in the protease locus that will enable us to characterize posttranslational processing of PrcA and PrtP, expression of PrcB, localization of protease complex proteins and assembly of the active protease complex. Aim 2: Determine the role of Msp in biosynthesis of protease complex proteins, and the role of the protease in oligomerization of Msp. We will characterize interactions between Msp and protease operon proteins that influence expression and activity of each outer membrane complex. Aim 3: Characterize interactions of Td outer membrane components with epithelial cells. To further studies of host cell responses to Td challenge, we will characterize interactions between Msp and a putative receptor identified on epithelial cells and characterize early events in epithelial cell innate responses to the PrtP protease complex and Msp, with the goal of differentiating between roles of Msp and the protease complex in cellular responses to Td. This project, involving genetic and biochemical analyses of Td outer membrane complex expression and analysis of Td effects on host cells, will contribute to molecular analysis of microbe-host interactions in the oral environment. Our laboratory is uniquely positioned to conduct these studies. Completion of these Aims will contribute to both basic knowledge of spirochete molecular biology and to understanding of microbe-host interactions in chronic infections such as periodontal diseases. Public Health Significance: This project will provide information on how a key bacterium involved in initiation periodontal disease interacts with human cells. The studies will first characterize the process of protein complex assembly on the bacterial surface. Then we will determine which specific proteins on human cells these complexes bind to, and then assay cellular responses to bacterial protein complexes binding to cell surface receptors.